The invention relates to methods for identifying human cells useful in endogenous gene activation, in order to produce human proteins. The invention also involves processes for manufacture of proteins, such as human proteins in cells identified in this manner, as well as the isolated cells so identified.
The production of human proteins by endogenous gene activation in a human cell line is known. See, e.g., WO 93/09222, WO 94/12650 and WO 95/31560 for example, describing the production of human erythropoietin and other human proteins in human cell lines by endogenous gene activation.
These references do not advise, however, as to what criteria have to be observed when selecting cells used to produce human proteins. In fact, the methods described in these references do not ensure high yield and contaminant free production of human protein. In fact, only low yields of human proteins are achieved following the above cited references.
As noted, supra, human erythropoietin is described in these references as a protein, the production of which is desired. A discussion of erythropoietin and the art relating to its production is set forth here, although it is to be borne in mind that erythropoietin production is simply exemplary of the invention, which is in no way limited to this protein.
Erythropoietin (xe2x80x9cEPOxe2x80x9d hereafter) is a glycoprotein which stimulates the production of red blood cells. EPO is present only in very low concentrations in the blood plasma of healthy persons, so it is not possible to prepare large amounts via purification of plasma. EP-B-0148 605 and EP-B-0205 564, incorporated by reference, describe the production of recombinant human EPO in Chinese Hamster ovary, or xe2x80x9cCHOxe2x80x9d cells. The EPO described in EP-B-0148 605 has a higher molecular weight than EPO purified from urine and is not O-glycosylated. The EPO from CHO cells described in EP-B-0 205 564 is available in large amounts and in pure form, but it is derived from non-human cells. Further, the production yield of CHO cells is also often relatively limited.
As alluded to pa, it is known that human EPO (xe2x80x9chEPOxe2x80x9d) can be isolated from the urine of patients with aplastic anemia (Miyake et al., J. Biol. Chem. 252 (1977), 5558-5564). A seven-step process is disclosed in this reference, which involves, inter alia, ion exchange chromatography, ethanol precipitation, gel filtration and adsorption chromatography. In this process an EPO preparation with a specific activity of ca. 70,000 U/mg protein is obtained in a 21% yield. The disadvantages of this process and other processes for isolating urinary EPO include obtaining the starting material in adequate amounts and with a reproducible quality. Furthermore, the purification of hEPO from urine is difficult and even a purified product is not free of urinary impurities.
GB-A-2085 887, incorporated by reference, describes a process for the production of human lymphoblastoid cells which are able to produce EPO in small amounts. It is not possible to economically produce EPO of the desired quality using the human lymphoblastoid cells disclosed herein.
WO 91/06667 as noted supra, describes a process for the recombinant production of EPO. In this process the endogenous EPO gene is operatively linked to a viral promoter in a first process step by homologous recombination, in primary human embryonic kidney cells. The recombined DNA is then isolated from these cells. In a second step, the isolated DNA is transformed into CHO cells, and the expression of EPO in these cells is analyzed. There is no indication that it is possible to produce EPO in human cells.
WO 93/09222 describes the production of EPO in human cells. In this process relatively high levels of EPO production, i.e., up to 960,620 mU/106 cells/24 hours is achieved using human fibroblasts which have been transfected with a vector containing the complete EPO gene. These transfected cells contain an exogenous EPO gene which is not located at the correct EPO gene locus, leading to problems with respect to the stability of the cell line. The reference does not discuss constitutive EPO production. Moreover, there is also no information about whether the EPO produced is of sufficient quality for, e.g., pharmaceutical use.
Activation of the endogenous EPO gene in human HT1080 cells is also described in this reference, but production of only 2,500 mU/106 cells/24 hours (corresponding to ca. 16 ng/106 cells/24 hours) is found. Such low production levels are unsuitable for economic production of a pharmaceutical preparation.
WO 94/12650 and WO 95/31560, incorporated by reference, describe that a human cell with an endogenous EPO gene activated by a viral promoter is capable, after amplification of the endogenous EPO gene, of producing EPO in an amount of up to ca. 100,000 mU/106 cells/24 hours (corresponding to ca. 0.6 xcexcg/106 cells/24 hours). Even this amount is still not sufficient for the economic production of a pharmaceutical preparation.
As indicated, supra, the cells and cell lines disclosed in the literature relating to endogenous gene activation, while useful, are by no means totally satisfactory. It has now been found, however, that it is possible to identify and to isolate cells and cell lines which will be useful in high yield production of proteins, following endogenous gene activation via, e.g., homologous recombination. Hence, one aspect of the invention is a method for identifying such cells and cell lines. A second feature of the invention are the cells and cell lines so identified. Yet a third feature of the invention is the use of the cells and cell lines for the production of proteins using these cells and cell lines. How these and other aspects of the invention are achieved will be seen from the disclosure which follows.